Contactin-associated protein-like 2 (CNTNAP2) mutations impair the essential α-secretase cleavages, leading to autism-like phenotypes

Mutations in the Contactin-associated protein-like 2 (CNTNAP2) gene are associated with autism spectrum disorder (ASD), and ectodomain shedding of the CNTNAP2 protein plays a role in its function. However, key enzymes involved in the C-terminal cleavage of CNTNAP2 remain largely unknown, and the effect of ASD-associated mutations on this process and its role in ASD pathogenesis remain elusive. In this report we showed that CNTNAP2 undergoes sequential cleavages by furin, ADAM10/17-dependent α-secretase and presenilin-dependent γ-secretase. We identified that the cleavage sites of ADAM10 and ADAM17 in CNTNAP2 locate at its C-terminal residue I79 and L96, and the main α-cleavage product C79 by ADAM10 is required for the subsequent γ-secretase cleavage to generate CNTNAP2 intracellular domain (CICD). ASD-associated CNTNAP2 mutations impair the α-cleavage to generate C79, and the inhibition leads to ASD-like repetitive and social behavior abnormalities in the Cntnap2-I1254T knock-in mice. Finally, exogenous expression of C79 improves autism-like phenotypes in the Cntnap2-I1254T knock-in and Cntnap2−/− knockout mice. This data demonstrates that the α-secretase is essential for CNTNAP2 processing and its function. Our study indicates that inhibition of the cleavage by pathogenic mutations underlies ASD pathogenesis, and upregulation of its C-terminal fragments could have therapeutical potentials for ASD treatment.


INTRODUCTION
Autism is a common neurodevelopmental disorder characterized by deficits in communication, social interaction, and repetitive/ restrictive behaviors and interests.Contactin-associated protein-like 2 (CNTNAP2) gene has been associated with autism, as both rare and common variations have been identified in patients with autism spectrum disorder (ASD). 1,25][6][7][8] However, the mechanism underlying the role of CNTNAP2 in autism remains elusive.
0][11] The gene encoding for the human CNTNAP2 protein locates in chromosome 7, and we reported that intermittent hypoxia treatment enhanced CNTNAP2 transcription to upregulate its protein expression. 12The CNTNAP2 protein belongs to the CNTNAP subfamily of the Neurexin family and functions as a cell adhesion molecule. 11,13It was initially found in a complex with voltage-gated potassium channel Kv1 and transient axonal glycoprotein-1 (TAG-1) in the juxtaparanodal region. 14,15CNTNAP2 is also located at pre-and post-synaptic sites, 16,17 and it plays an essential function in axonal growth, 18 dendritic arborization, 19 and spine development. 17,20Our recent study has demonstrated that CNTNAP2 full-length protein and its C-terminal fragments (CTF) have a half-life of approximately 3-4 h.Its degradation is dependent on both ubiquitin-proteasome system (UPS) and the macroautophagy-lysosome pathway, while the latter is the more common way for CNTNAP2 degradation. 21ome synaptic molecules, such as neurexin 3β and neuroligin-1, undergo ectodomain shedding. 22,235][26] Both APP and Notch can be cleaved directly by α-secretase and then γ-secretase.Three members of the A Disintegrin And Metalloproteinase (ADAM) family have been identified as candidates for the α-secretase: ADAM10, ADAM17 and ADAM9.While ADAM10 is widely expressed in the central nervous system, ADAM17 is more restrictedly expressed in the hippocampus.Both ADAM10 and ADAM17 are cell membrane-bound proteases that cleave substrates including growth factors, receptors and cytokines primarily at the extracellular sites close to the cell membrane.APP is a single-pass transmembrane protein with a large extracellular N-terminal domain, and a shorter cytoplasmic C-terminal tail.Under physiological conditions, APP is predominantly cleaved by α-secretase between Lys-16 and Leu-17 within the amyloid β protein (Aβ) sequence to create sAPPα and CTFα C83, and the resulting C83 is subsequently cleaved by the γ-secretase to yield P3α and CTFγ. 27In the non-amyloidogenic pathway, ADAM10 is the constitutive αsecretase, while ADAM17 functions in the regulated α-secretase cleavage of APP.In the Golgi, Notch is cleaved by furin (S1 cleavage) and glycosylated by O-fucosyltransferase.The processed Notch receptor is then transported to the cell membrane, where it binds to the Delta ligand.Upon ligand binding, the Notch extracellular domain is cleaved away by ADAM10/ ADAM17-dependent αsecretase (S2 cleavage).The membrane-bound fragment is further cleaved by γ-secretase (S3 cleavage) to release the Notch intracellular domain (NICD).The proteolytic processing of Notch to generate NICD is critical for normal development. 25γ-secretase is an enzyme complex that contains presenilins (PS) as its core component.This high molecular weight complex also requires the presence of nicastrin, anterior pharynx-defective 1 (APH-1), and PEN-2 for its enzymatic activity. 28PS1 and PS2 are homologous with 67% identical sequences.They are both ubiquitously expressed in human and mouse tissue, while PS1 mRNA levels are significantly higher in developing brains.It has been confirmed that a total elimination of γ-secretase activity in the PS1/PS2 double knockout cells established the central role of presenilins in the γ-secretase complex.PS1-deficient neurons had a markedly reduced APP γ-secretase cleavage and Aβ generation, arguing for PS1 as the principal component of the γ-secretase complex. 29PS1 is also required for the γ-cleavage of Notch. 25ike APP and Notch, CNTNAP2 has a long extracellular N-terminal and a short intracellular C-terminal.CNTNAP2 is cleaved by matrix metalloproteinase 9 (MMP9), and the shed ectodomain regulates Ca 2+ homeostasis and neuronal network synchrony. 30Recently, we found that CNTNAP2 is cleaved by presenilins-dependent γ-secretase to produce the CNTNAP2 intracellular domain (CICD), and expression of the CICD improves autism-like behaviors. 31Our current study reveals that in addition to its γ-secretase cleavage, CNTNAP2 undergoes complex proteolytic processing by furin and a disintegrin and metalloproteinase (ADAM)10/17-dependent α-secretase.The main α-cleavage product C79 by ADAM10 is required for the subsequent γ-secretase cleavage to generate the CICD.Furthermore, ASD-associated pathogenic CNTNAP2 mutations inhibited the α-cleavage, leading to autism-like phenotypes in vivo, whereas exogenous expression of the α-cleavage product C79 improves autism-like phenotypes in the Cntnap2 -I1254T knock-in and Cntnap2 −/− knockout mice.

Identification of α-cleavage sites on CNTNAP2
To identify the α-secretase cleavage sites, plasmids expressing the last 178, 108, and 91 aa of CNTNAP2 C-terminus were co-transfected with ADAM10 or ADAM17.CTFα1 and CTFα2 were generated from both 178 and 108 constructs while only CTFα1 was produced from the 91 construct, indicating that the α-secretase site generating CTFα2 is within the last 108 aa, and the CTFα1 is within the last 90 aa (Supplementary Fig. 3a).Next, Mass Spectrometry and N-terminal sequencing were performed to identify the cleavage sites.Synthetic peptide 1 (C-terminal 108-84 aa of CNTNAP2) and peptide 2 (Cterminal 96-72 aa) were treated with the recombinant human ADAM17 protein, and Mass Spectrometry results showed that ADAM17 cleaved peptide 1 between H97 and L96 and peptide 2 between A80 and I79 (Fig. 2a).N-terminal sequencing also identified that CTFα1 started from IRNGV, and CTFα2 started from LDSAS (Fig. 2b, Supplementary Fig. 3b).In addition, we generated truncated CNTNAP2 C-terminal protein ladders C79 and C96, which perfectly matched the size of CTFα1 and CTFα2 (Fig. 2c).These results clearly demonstrated that CNTNAP2 is cleaved by αsecretase primarily at I79 to generate C79 (CTFα1), and less at L96 to yield C96 (CTFα2), respectively.
Taken together, these data indicated that CNTNAP2 undergoes complex sequential cleavages by furin and two α-secretases: ADAM10 and ADAM17.ADAM10 and ADAM17 share the same αcleavage sites at L96 and I79 but show different site preferences, with ADAM17 predominantly at L96 and ADAM10 primarily at I79, resulting in C79 as the main CTF cleavage product of CNTNAP2 through sequential cleavages (Fig. 3g).
Fig. 2 Identification of α-secretase cleavage sites on CNTNAP2.a Peptide in vitro cleavage.Two peptides were incubated in the assay buffer with or without ADAM17 at 37 °C for 24 h and then were sent to Mass Spectrometry analysis.The extracted ion chromatogram showed two intact peptides (top) and cleaved complementary fragments (bottom).Peptide 1 (M108-G84) was cleaved by ADAM17 at 97H/L96.Peptide 2 (L96-N72) was cleaved at 80 A/79I.b N-terminal sequencing results identified the first 5 amino acids (aa) of CTFα1 as IRNGV and the first 5 aa of CTFα2 as LDSAS.The bottom sequence shows the last 108 to 72 aa of CNTNAP2 from the C-terminus and α-secretase cleavage sites at L96 and I79.c Protein ladders of the C-terminal CNTNAP2.C79 corresponded to the size of CTFα1, and C96 showed the same size as CTFα2.d, e Mutations at the α-secretase cleavage sites L96 and I79 affected CNTNAP2's cleavage.Wildtype (WT) or mutant plasmids were cotransfected with empty vector (EV), ADAM10 (AD10), or ADAM17 (AD17) into HEK cells.Alterations in the migration rate were observed in constructs containing D98R.Two upper/lower blots show samples from the same experiment.The parallel blots were processed in the same electrophoresis chamber and scanned together simultaneously.n = 3 independent experiments, two-way ANOVA followed by Dunnett's multiple comparisons test (compare ADAM10 and ADAM17 with Control for each plasmid), row factor = mutation, column factor = ADAM10/ 17 overexpression.*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.All the results are expressed as mean ± SEM Cntnap2 -I1254T mice exhibit autism-like phenotypes and the C79 improves the phenotypes in vivo To examine the effect of the essential α-secretase cleavage of CNTNAP2 on autism-like phenotypic behaviors, we generated mutant I1254T knock-in mice (Cntnap2 -I1254T ) using the CRISPR/ Cas9 technique to express the I1254T mutant CNTNAP2 protein, corresponding to the pathogenic I1253T mutation in ASD patients (Fig. 5a).All mice were bred through crossing heterozygous Cntnap2 -I1254T and further confirmed by genotype sequencing.The pups were born at a Mendelian ratio, and there was no gender difference of pups (Supplementary Table 2 and Supplementary Fig. 5).Cntnap2 -I1254T mice grew as well as control mice during the 8 weeks of study period.Body weight was monitored from 3 to 8 weeks of age, and there was no difference in body weight between the mutant Cntnap2 -I1254T mice and control WT mice (Fig. 5b).
The mice were tested with the isolation-induced ultrasonic vocalizations (UsVs) at the ages of 3, 6 and 12 days, in which the distress calls emitted by separated pups and the infant-mother vocal communications were considered as ASD-like behavior. 3,32ntnap2 -I1254T mice emitted a significantly lower number of distress calls than WT mice at different ages (Fig. 5c).In the Juvenile Play test where the social interaction and repetitive behaviors between different pairs of unfamiliar mice was recorded, the Cntnap2 -I1254T mice showed increased repetitive behaviors like grooming (Fig. 5d).
To confirm the autism-like behaviors was due to the impaired αcleavage of CNTNAP2 at ADAM10 site, the adeno-associated virus (AAV) vector expressing CNTNAP2-C79 protein (AAV-C79) and AAV-EGFP (as the control) were generated and injected into the medial prefrontal cortex (mPFC) of the mutant (Cntnap2 -I1254T ) and CNTNAP2-KO mice (Cntnap2 −/− ) at the age of 3 weeks (Supplementary Fig. 6a).AAV-C79 injection significantly increased the C79 protein level in mPFC (Supplementary Fig. 6b).In an open-field test, Cntnap2 -I1254T mice displayed locomotor activity similar to that of the WT mice (Supplementary Fig. 7a), while the Cntnap2 −/− mice exhibited hyper locomotion compared with the WT mice (Supplementary Fig. 7b).In addition, Cntnap2 -I1254T mice injected with AAV-EGFP displayed an increased number of no alterations, representing an ASD-like repetitive behavior (Fig. 5e).In the threechamber social interaction test, all mice showed no preference for each chamber in the habituation phase (Supplementary Fig. 8a, b), and Cntnap2 -I1254T mice did not show a significant social preference for strangers when compared to the highly socialinteractive WT mice (Fig. 5f).However, the expression of C79 significantly reduced the number of no alterations of Cntnap2 -I1254T mice in T maze test (Fig. 5e) and increased their social interactions with unfamiliar mice (Fig. 5f) at the age of 7 weeks, indicating that C79 expression improved autism-like phenotypes in the mutant mice.Similarly, C79 expression also rescued the deficiency in repetitive and social behaviors of Cntnap2 −/− mice (Fig. 5g-i).Taken together, these data demonstrate that the mutation of α-cleavage site in CNTNAP2 leads to autism-like repetitive and social behavior abnormalities, and restoring α-cleavage product C79 expression improves autismlike phenotypes.
The current study demonstrates that CNTNAP2-C79 generated by α-secretase cleavage is absolutely required for the subsequent γ-secretase cleavage to yield the CNTNAP2-CICD.We recently reported that CICD enters the nucleus and functions as a transcription factor to improve autism-related behaviors. 31Therefore, disruptions in the α-cleavage of CNTNAP2, such as by pathogenic mutations shown in Fig. 4, may affect the downstream CNTNAP2 signaling pathway.
ADAM10 is widely expressed in the brain and functions as a major protease in the central nervous system. 39It is synthesized in the rough endoplasmic reticulum (ER) and undergoes maturation as it is transported through the Golgi apparatus.During maturation, the prodomain of ADAM10, which keeps the enzyme in an inactive state, is removed.This removal of the prodomain is a crucial step in activating ADAM10 and allowing it to exert its enzymatic functions.As a prominent α-secretase enzyme, it is primarily recognized for its involvement in APP processing to regulate the production of Aβ.In the context of AD, the activation of ADAM10 has also been shown to elevate the levels of soluble triggering receptor expressed on myeloid cells-2 (sTREM2) by influencing the extracellular domain of TREM2.The sTREM2 is known to possess neuroprotective properties as it enhances the degradation of Aβ plaques. 40ADAM10 also plays a significant role in the shedding of proteins that are crucial for brain development, including cadherins, ephrins, and Notch receptors.Notch receptors are key regulators of cell proliferation, cell fate decisions and differentiation.ADAM10 is involved in the proteolytic cleavage of Notch receptors, which is a critical step in the activation of Notch signaling and influences cell fate decisions during neurogenesis and brain development.Adam10-KO mice die at embryonic day 9.5 due to disruptions of the Notch signaling. 41Both conditional Adam10-KO mice 42,43 and Cntnap2-KO mice 3,44 are viable but have seizures, learning deficits, neuronal migration abnormalities, and Fig. 3 Sequential cleavages of furin, αand γ-secretase.a Western blot of control (Vector), ADAM10 overexpressing, and ADAM17 overexpressing HEK cells.Exposure 1 was used to analyze CTFf in the ADAM17 overexpressing cells and C96 in the ADAM10 overexpressing cells; exposure 2 was used for assessing C79 and C96 in the ADAM17 overexpressing cells.TAPI-1 (10 μM) is an ADAM17-specific inhibitor; FI-2 (20 μM) stands for Furin inhibitor-2.b Quantification of the dynamic changes of mature CNTNAP2 and CTF levels in the ADAM10/ADAM17 transfected cells upon inhibitor treatments in (a).n = 3 independent experiments, ordinary one-way ANOVA followed by Tukey's multiple comparisons test, *p < 0.05; **p < 0.01; ***p < 0.001.P values stand for comparisons with the Control group unless noted by the brackets.c, d Dynamic changes of CTFs in the furin transfected cells, showing the furin-ADAM17 cleavages.n = 3 independent experiments, ordinary one-way ANOVA followed by Tukey's multiple comparisons test, *p < 0.05; **p < 0.01; ***p < 0.001.P values stand for comparisons with the Control group unless noted by the brackets.e Western Blot of control (Vector), ADAM10 overexpressing, and ADAM17 overexpressing HEK cells.GSI is γ-secretase inhibitor L-685,458 (20 nM).f Quantification of the sequential cleavage by αand γ-secretase in (e).n = 3 independent experiments, unpaired t-test, *p < 0.05, **p < 0.01.g Schematic of CNTNAP2 processing.Mature CNTNAP2 undergoes sequential cleavages by furin and α-, γ-secretase.Furin cleaves CNTNAP2 to generate CTFf, which is further processed by α-secretase.α-secretase ADAM10 and ADAM17 share the same cutting sites but have different site preferences.ADAM10 primarily cleaves at I79 to produce the predominant C79, and ADAM17 preferably cleaves at L96 to produce the weaker C96.C96 is subsequently processed by α-secretase into C79.γ-secretase further cleaves C79 at L53 within the transmembrane domain to generate CICD.All the results are expressed as mean ± SEM aberrant spine morphology, implicating the significance of CNTNAP2 as a critical substrate cleaved by ADAM10.Notably, we found that the CNTNAP2-CICD or C79 is sufficient to rescue the behavioral deficits in the Cntnap2 −/− knockout mouse, suggesting the cleaved C79 is the key molecule for CNTNAP2's physiological functions.Consistent with the pathogenic effect of I1253T mutation in ASD patients, Cntnap2 -I1254T knock-in mice displayed characteristic autism-like phenotypes that can be rescued by Post-translational modifications play a crucial role in determining and regulating a protein's structure, localization and functions.APP undergoes N-and O-glycosylation, phosphorylation, sulfation, palmitoylation, ubiquitination and sumoylation following its synthesis.It is believed that these post-translational modifications are required for the cleavage of APP within an intracellular secretory pathway. 45APP modifications also play a crucial role in its trafficking, which is mutually regulated and collectively contributes to the regulation of APP processing and Aβ generation. 45Similar to APP processing, our study has revealed that the cleavage of CNTNAP2 by the proteases occurs only in its mature form.CNTNAP2 undergoes N-linked glycosylation during its maturation. 10,11,18Disruptions in glycosylation could affect CNTNAP2 trafficking, localization, interaction, and degradation, particularly given that mature and immature forms of CNTNAP2 are degraded in different degradation pathways. 21We and others found that ASD pathogenic mutations I869T, R1119H, and D1129H, have reduced mature/immature ratios and trafficking deficits, leading to retention in the endoplasmic reticulum. 18As expected, our study found that these mutations affected αsecretase cleavage and reduced C79 generation.Future studies are warranted to further examine CNTNAP2's post-translational modifications, which could be critical for CNTNAP2's proteolytic processing.Although G731S and R906H showed relatively normal maturation rates, ADAM10-dependent α-secretase cleavage was largely reduced, resulting in less C79 generation.The impaired cleavage is most prominent in I1253T, which resides precisely at the α-cleavage site.Although pathogenic mutations may contribute to ASD pathogenesis by different mechanisms, one common consequence is the reduced C79 level.
CNTNAP2 is processed by MMP9, and the released ectodomain regulates Ca 2+ homeostasis and reduces neuronal network synchrony. 30MMPs and ADAMs, both belonging to metzincin metalloproteinases, are widely expressed in the nervous system 46 and sometimes share substrates.For instance, syndecan-1 and syndecan-4 are cleaved by both MMP9 and ADAM17 close to the cell membrane. 47,48Our study clearly identified two α-cleavage sites by ADAM10 and ADAM17, and our results from the KO cell lines unequivocally demonstrated that CNTNAP2 is mainly cleaved by ADAM10.Additionally, this study is the first to show the Fig. 5 Impairment of α-secretase cleavage resulted in repetitive and social behavior abnormalities in mice.a Schematic representation of CRISPR/Cas9 knock-in method used to generate mutant I1254T mice by mutating "ATA" (Ile) to "ACG" (Thr) at the major α-cleavage site of CNTNAP2.b Body weight record of WT and I1254T mice.The weight of both mice was recorded daily, and no significant difference was found between mutants and WT mice (WT, n = 15; I1254T, n = 15).c Isolation-induced ultrasonic vocalizations (UsVs) test.The numbers of distress calls from infants of WT and mutants were detected at the age of P3, P6 and P12 (WT, n = 13; I1254T, n = 13).d Juvenile play test.Time involved in social interaction, as well as repetitive behaviors like grooming in WT and I1254T mutant mice were measured at the age of P21 when interacting with unfamiliar mice (n = 10 per group: male = 5, female = 5).e, f T maze and three chamber sociability tests performed at 7-week-old WT and mutants.I1254T mutation at the α-cleavage site increased repetitive behavior abnormalities in T maze (e) and decreased social interactions (f) in I1254T mutants, and C79 overexpression rescued the ASD-like behaviors (n = 12 per group: male = 6, female = 6).g-i Social and repetitive behavior tests in Cntnap2 −/− mice.C79 overexpression rescued the social interactions (g, i) and repetitive behaviors (h) in Cntnap2 −/− mice (n = 11: male = 6, female = 5 in WT and KO groups; n = 12: male = 6, female = 6 in KO + C79 group).Statistical significance was assessed by either one-or two-way ANOVA followed by Turkey or Bonferroni's test.*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.All the results are expressed as mean ± SEM complete CTF pattern of CNTNAP2.Our finding that CNTNAP2 undergoes complex proteolytic processing provides novel clues for in-depth studies of the CNTNAP2 signaling pathway.Future studies are warranted to reveal the functions of cleaved ectodomains and CTFs.Here we show that C79 generated by α-secretase is a key molecule of CNTNAP2 signaling, the disruption of which may contribute to ASD pathogenesis.Furthermore, we found that restoring C79 expression improves autism-like phenotypes in the CNTNAP2 -I1254T mutant mice and CNTNAP2 −/− knockout mice.Therefore, replenishment of CNTNAP2-C79 or CNTNAP2-CICD to the physiological level could be a potential avenue to treat ASD.
Immunoblot analysis Cells were lysed in RIPA lysis buffer supplemented with the cOmplete Protease Inhibitor Cocktail (Roche).Cell lysates were resolved on 7.5% Tris-glycine or 16% Tris-tricine (for CTF) SDS-PAGE and transferred to nitrocellulose membranes.Membranes were blocked in 5% non-fat milk in phosphate-buffered saline (PBS) for 1 h at room temperature and then blotted with primary antibodies overnight at 4 °C with shaking.On the next day, membranes were blotted with IRDyeTM 680-labeled (1: 10000, LI-COR Biosciences Cat# 926-68070, RRID: AB_10956588) or IRDyeTM 800CW-labeled (1: 10000, LI-COR Biosciences Cat# 926-32211, RRID: AB_621843) secondary antibodies for 1 h at room temperature and then visualized using the LI-COR system.The following rabbit antibodies were used: anti-FLAG (1: 2000, Abcam Cat# ab1162, RRID: AB_298215) was used to detect CTF with Flag tag; N63 (1:1000, targeting 63-95 aa of CNTNAP2 at the N-terminal) and C22 (1:1000, targeting the last 22 aa of CNTNAP2 at the Cterminal), generated by our lab, 21 were used to detect CNTNAP2 full-length and CTF; anti-ADAM10 (1:1000, Abcam Cat# ab124695, RRID: AB_10972023) and anti-ADAM17 (1:1000, Santa Cruz Biotechnology Cat# sc-390859) were used to detect endogenous ADAM10 and ADAM17; anti-GFP (1:1000, Santa Cruz Biotechnology Cat# sc-8334, RRID: AB_641123) was used to detect GFP.The following mouse antibodies were used: anti-HA (1:50, 12CA3, generated in our lab) was used to detect CNTNAP2 full-length with HA tag; anti-myc (1:20, 9E10) was used to detect transfected ADAM10, ADAM17, and furin with C-terminal myc tag; anti-Actin (1:50000, Sigma-Aldrich Cat# A1978, RRID: AB_ 476692) was applied for Actin detection.Immunocytochemistry HEK and N2a cells were transfected and then plated on coverslips pre-coated with Poly-D-Lysine (Sigma-Aldrich Cat# P7886).24 h later, cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature and then washed 3 times with PBS.After blocking with 5% Bovine Serum Albumin (BSA) in PBS for 1 h at room temperature, cells were incubated with primary antibodies overnight at 4 °C in a humidity chamber.On the next day, cells were washed 3 times with PBS and then incubated with secondary antibodies for 1 h at room temperature in a humidity chamber protected from the light.After 3 washes with PBS, coverslips were mounted using Fluoromount-G with DAPI (Invitrogen Cat# 00-4959-52) and air-dried in the dark.CNTNAP2 was detected by anti-CNTNAP2 targeting extracellular 1001-1142 aa (1: 200, Sigma-Aldrich Cat# HPA002739, RRID: AB_1078545) and 488-Donkey anti-Rabbit IgG (1: 500, Thermo Fisher Scientific Cat# A-21206, RRID: AB_2535792).Myc-tagged ADAM10, ADAM17, and furin were detected by anti-myc (1:200, 9E10, generated in our lab) and 568-Goat anti-Mouse IgG (1: 500, Thermo Fisher Scientific Cat# A-11031, RRID: AB_144696).Antibodies were prepared with 1% BSA in PBS.The staining results were viewed under the 63x oil lens of the Zeiss fluorescence microscope.Images taken under the Apotome mode were used to reflect the membrane expression of CNTNAP2 in Fig. 1g.Corresponding conventional fluorescent images (Supplementary Fig. 2) were quantified using Zeiss software to investigate the cleavage.10 cells for each transfection group were randomly chosen from two independent experiments (5 cells from each).The exposure time for each channel in HEK and N2a cells was as follows: DAPI 8 ms, EGFP (green) 15 ms, and AF568 (red) 60 ms.The area of individual cell was outlined by the Spline Contour graphic.Then, the mean intensity value for the outlined cell area was automatically produced in the Measure tab in Zeiss software.The average intensity of 10 cells from the vector group was used to normalize the value across all transfection groups in percentage.
Immunoprecipitation and N-terminal sequencing 30 × 10 cm plates of HEK cells were co-transfected with pRK5-CNTNAP2 and pRK5M-ADAM17 and then treated with 5 μM MG132 (Sigma-Aldrich Cat# C2211) and 20 nM γ-secretase inhibitor L-685,458 for 16 h before harvested to increase CTF.Cells were lysed using RIPA buffer and precleared with Sepharose 4B (Sigma-Aldrich Cat# 4B200) for 1 h at 4 °C.Precleared lysates were incubated with anti-FLAG M2 affinity gel (Sigma-Aldrich Cat# A2220, RRID: AB_10063035) overnight at 4 °C with rotating.On the next day, unbound proteins were washed out, and precipitated proteins were eluted via boiling in 1 x sample buffer.Eluted proteins were resolved on 16% Tris-tricine SDS-PAGE and transferred to PVDF membranes.Membranes were stained with Coomassie blue to visualize the precipitated CTF and then sent for N-terminal sequencing (The Protein Facility of the Iowa State University).

Mice
Cntnap2 mutant and wild-type (WT) mice were generated through crossing mutant heterozygous Cntnap2 mutant mice, and offspring were born with the expected Mendelian frequencies.The genotypes of the mice were confirmed by sequencing the DNA extracted from tail tissues with forward primer CNTNAP2 5′-TGGATACTACAAGACAATAGCAAG and reverse primer CNTNAP2 5′-ACCTTGATGAGGCTATAATTGAAC.3-4 mice of the same sex were raised in each cage with 12-h light and dark cycle, and water and food were available at all day.For each behavioral test, 10-15 mice injected with either AAV-EGFP or AAV-C79 were included.All behavioral tests were performed and analyzed with the ANY-maze automated system.Ultrasonic Vocalizations were analyzed with Sonotrack sound analysis and synthesis software for laboratory animals.All procedures regarding the care and use of animals were approved by the Ethics Committee of Wenzhou Medical University.All animal procedures were performed in accordance with the Wenzhou Medical University Animal Research Center protocols.

Ultrasonic Vocalization (UsV) Test
To detect distress calls emitted by separated infants, pups at different ages of 3, 6, and 12 days were transferred into 400 × 300 × 220 mm cardboard boxes that were outfitted with UsV recording equipment for 5 min.The room temperature was kept at 21 °C to prevent temperature-related variable factors.The total number of distress calls within 5 min was recorded by Sonotrack software.

Juvenile play test
The animals were allowed to habituate for a minimum of 1 h prior to the start of behavior test.Mice aged P21 were placed in a new cage and kept for 10 min with a genotype-and sex-matched stranger mouse.The mice performing social interactions such as nose-to-nose sniffing and nose-to-anal sniffing, as well as repetitive behaviors such as grooming were measured by human observer. 3,32maze spontaneous alternation In brief, 7-week-old mice were given proper acclimatization in the test room for a minimum of 1 h.Then, mice were situated at the base of a T-maze equipment to explore either the right or left arm of the maze for 10 consecutive trials.The number of no spontaneous alternations of mice were counted and recorded by the observer.
Reciprocal social interaction As previously described, 50 7-week old mice were tested in this experiment.After 1 h of acclimatization, the mice were habituated in a social box for 10 min.Then, an unfamiliar mouse of the same genotype, age and sex was introduced into the social box, and their interactions were recorded by video for 10 min.The interactions, including nose-to-nose, nose-to-genital communication, and body contacts, were measured.
Open field test Briefly, the whole experiment took place in a square arena (50 × 50 cm), which was mounted within specially designed sound attenuating shells constructed of polypropylene, regular and expanded PVC.All mice were placed in the center of the open field arena and allowed to freely move for 15 min while being tracked by an automated tracking system.
Three chamber social interaction test The social interaction test was performed as previously described. 3,7Briefly, after 1-h habituation, mice were placed in a clear Plexiglas box to explore all three divided chambers for 10 min to test locomotor activity.Then the mice were allowed to freely interact with either an empty wire cup (positioned in one side chamber) or with a wire cup with a stranger mouse inside (positioned in the opposite side chamber).The time of social interaction was measured and analyzed with an ANY-maze automated system.

Statistics
All the results are expressed as mean ± standard error of the mean (SEM).Replicate numbers of independent experiments, cells, or animals are noted in the figure legends.GraphPad Prism was used for statistical analyses and for creating quantification figures.Comparison between two groups was analyzed by the 2-tailed Student's t-test.Multiple comparisons were analyzed by one-way or two-way ANOVA followed by post hoc multiple comparisons test.P < 0.05 was considered statistically significant.